Reagents and kits for detection of influenza virus and the like

ABSTRACT

The present invention relates to reagents and methods for influenza virus detection. These reagents and methods disclosed in the present invention enable simple, rapid, specific and sensitive detection of influenza virus types A and B. These reagents are N-acetylneuraminic acid-firefly luciferin conjugates which can be cleaved by influenza virus neuraminidase.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application No.60/930,207, filed May 15, 2007, and 60/999,166, filed on Oct. 16, 2007.The entire disclosure of the prior application is considered to be partof the disclosure of the instant application and is hereby incorporatedby reference.

TECHNICAL FIELD

The present invention relates to influenza virus detection reagents andkits which use a group of chemicals that can be used for detection ofthe neuraminidase activity as a method for the detection of influenzaviruses present in a sample. The chemicals are conjugates betweenN-acetylneuraminic acid (sialic acid) or its derivatives and fireflyluciferin, which are linked together through a glycoside bond via an —OHgroup on the sugar ring of the N-acetylneuraminic acid, preferably the2′ position.

The present invention further relates to reagents and test kits for thedetection of other enzymes, pathogens or diseases using fireflyluciferin containing conjugates as substrates.

BACKGROUND OF THE INVENTION

Influenza is a constant and serious threat to public health. Each year,influenza epidemic leads to 200,000 hospitalizations and 36,000 deathsin the United States. Globally, influenza disease impacts every year upto 10% of the world's population—approximately 500 millions of thepeople.

An influenza pandemic could lead to far greater number of deaths andeconomic impact. The 1918 influenza pandemic, for example, killed 20-40million people in the world and more than 500,000 people in the UnitedStates. As medical science advances, there are several drugs that areavailable for treatment or prophylaxis of influenza A and B. However,the prerequisite for effective treatment or prevention control is rapid,sensitive and specific detection of the viruses at an early stage ofindividual infection or of an outbreak.

Conventional method for influenza virus detection involves first viralculture of a nasal wash, throat swab specimen, tracheal aspiratesecretions, bronchial lavages, or lung tissue. The virus in culturesusually is detected between day 2 and day 5 by performing hemabsorptiontest, which is normally followed by detection with an immunofluorescenceassay (IFA) using type specific antibodies. This test, while reliable,is not suitable for point-of-care use, which requires a test that canproduce a test result in a short period of time, e.g., less than 30minutes.

Consequently, faster influenza virus tests were developed to meet theneed of point-of-care use. These tests are of two types. One usesspecific antibody or antibodies to detect influenza proteins, orantigens. The other detects the activity of neuraminidase that ispresent in all type A and B influenza virus. Wile these tests cangenerally be completed in a short period of time, e.g., within 30minutes, they lack sufficient clinical sensitivity, which is generallyaround 70% when compared to virus culture methods. Lack of clinicalsensitivity for these rapid tests appears to be a result of lowanalytical sensitivity (or limit of detection), which is generally inthe range between 10⁴ and 10⁵ CEID₅₀ (Egg Infection Dose) or TCID₅₀(Tissue Culture infection Dose) units of influenza virus. The lack ofsensitivity seriously undermines the clinical usefulness of these tests.

More sensitive methods that involve the use of polymerase chain reaction(PCR) for detecting influenza virus nucleic acids have been described ina number of published articles. While these methods provide a moresensitive alternative, they generally have a number of shortcomings thatmake them less suitable for point-of-care use. For example, thesemethods generally demand complicate and lengthy sample preparation,which increases not only the cost, but also the time it takes tocomplete the test. These methods, particularly the so called real timePCR, also require expensive equipment and well-trained technicians,which cannot be afforded by most point-of-care facilities, e.g., thephysician's offices.

In an attempt to prepare a reagent that would provide better sensitivityfor influenza virus detection, a substrate for chemiluminescentinfluenza viral neuraminidase detection was synthesized (AnalyticalBiochemistry 2000; 280: 291-300). The substrate is a conjugate betweenN-acetyl-neuraminic acid and spiroadamantyl-1,2-dioxetane (hereinreferred to as dioxetane) through the 2′ position of the sialic acid.Cleavage of the linkage by neuraminidase releases the dioxetane, whichoxidizes to generate chemiluminescence under alkaline conditions.

Therefore, this substrate could be used for the detection ofneuraminidase activity, including that of influenza virus, as a means ofdetecting a pathogen such as influenza virus.

One shortcoming of the sialic acid-dioxetane substrate or methylatedform of this substrate is that the pH for the neuraminidase reaction andchemiluminescence reaction is significantly different (e.g., pH 6.5 vs.pH 11). This makes it necessary to separate these two reactions, whichincreases the complexity and cost of the assay.

Similar problems also exist for the detection of other pathogens anddiseases. Therefore, there is a need for reagents or kits that enablessimple, rapid and sensitive detection of influenza virus in a clinical,animal or environmental sample.

SUMMARY OF THE INVENTION

The present invention relates to reagents and methods for influenzavirus detection. These reagents and methods disclosed in the presentinvention enable simple, rapid, specific and sensitive detection ofinfluenza virus types A and B.

One embodiment of the current invention involves the use of an influenzavirus capture reagent, which is a solid support coated with an influenzavirus binding ligand. The capture reagent enables selective enrichmentof influenza virus from a sample. When used in an influenza detectionassay, the capture reagent improves the sensitivity of the assay byenriching the influenza virus in a sample. In addition, selectivebinding of the affinity ligand to influenza virus provides thespecificity of the assay even if the downstream detection process is notspecific for influenza virus.

Another embodiment of the present invention relates to a group ofchemicals that enable the detection of influenza virus by detecting theneuraminidase activity of influenza virus. These chemicals areconjugates between N-acetylneuraminic acid (sialic acid) or itsderivatives and firefly luciferin, which are linked together through aglycoside bond via an —OH group on the sugar ring of theN-acetylneuraminic acid. The preferred position on the sugar ring is the2′ position since this is the glycoside bond favored by influenzaneuraminidase. The conjugates used as the neuraminidase substrates canbe represented by the following formula (Formula I):

wherein R1, R2, R3 are each, independently of one another, hydrogen oralkyl groups comprising 1-5 carbon atoms, or a salt thereof.

In one embodiment the alkyl group is a straight chain or branched alkylgroup.

In a preferred embodiment, the alky group is methyl.

Suitable salts are those which are compatible with the assays of thisinvention, and include alkali metal (e.g., sodium, potassium, etc.),alkaline earth metal (e.g., calcium, magnesium, etc), ammonium, etcsalts.

In another embodiment at least one of R1, R2, or R3 is not hydrogen. Arepresentative example is shown in FIG. 4.

Components not specifically discussed herein are conventional in thefield of the assays of this invention, including chemicals compatiblewith such assays, e.g., buffers, solvents, etc.

When used for detection of influenza viral neuraminidase activity, theconjugate is incubated under appropriate conditions with a sample to betested. If the sample contains detectable amounts of influenza virus,the viral neuraminidase cleaves the conjugate and releases fireflyluciferin. A second reagent mix, which contains necessary reagents withthe exception of firefly luciferin for firefly luciferase catalyzedbiochemiluminescence reaction, is added to the neuraminidase reaction.Presence of free firefly luciferin enables the biochemiluminescencereaction, hence the production of light signal, which is indicative ofthe presence of influenza virus. The light signal can be detected with aluminometer, which is readily available from many commercial vendors(e.g., Promega). With this test design, a test kit contains at least aneuraminidase reaction reagent mix and a biochemiluminescence reactionmix. The detection procedure involves at least two major steps.

Yet another embodiment is combining the viral neuraminidase andluciferase-based biochemiluminescence reactions together in one assaystep such that the firefly luciferin cleaved by the viral neuraminidasefrom the conjugate is immediately consumed by luciferase to give rise tolight signal. Because the released firefly luciferin is immediatelymeasured as it is being generated by neuraminidase, this assay format isessentially a real time assay for neuraminidase activity. The real-timeassays are generally more quantitative and thus more appropriate forcertain applications that require quantitation capability of aneuraminidase assay such as the resistance testing for anti-influenzadrugs that target the neuraminidase. Combining the two reactions intoone assay step also simplifies the detection procedure, which makes itsuitable for point-of-care use. A test kit contains one key reagentcomponent, which is the master mix that contains all necessary reagentsfor both the neuraminidase and firefly luciferase reactions except forneuraminidase and firefly luciferin. In preferred embodiments, themaster mix is lyophilized for long term storage.

The invention provides methods for specific detection of influenza viralneuraminidase for diagnostic. In certain embodiments, inhibitors andantibodies are used to inhibit the neuraminidase activities fromundesired sources, e.g., a bacterial species. In other embodiments, thetest uses neuraminic acid-firefly luciferin conjugates, e.g., the 4,7methylated N-acetylneuraminic acid-firefly luciferin conjugate, whichfavors influenza viral neuraminidase and thereby improves the detectionspecificity.

The present invention also provides methods for detection of influenzavirus using a non-specific substrate. The undesired neuraminidaseactivity can be inhibited with polyclonal or monoclonal antibodies orwith small blocking molecules that are specific for the undesiredneuraminidase activity. Alternatively, a sample can be tested induplicate with two detection reagent mixes, one of which containsinfluenza viral neuraminidase specific inhibitor, e.g., oseltamivircarboxylate, while the other contains no inhibitor. Presence ofdetectable influenza virus in a sample is indicated if the detection mixwithout inhibitor is positive and the inhibitor-containing detection mixis negative. If both are positive, it indicates infection due to certainbacterial species or virus (e.g., parainfluenza virus).

In addition, the present invention provides similar methods for thedetection of other enzymes, which may be indicative of a pathogen ordisease. For example, sialidase (neuraminidase) is a marker forbacterial vaginosis. Thus, the N-acetylneuraminic acid-firefly luciferinsubstrate (FIG. 3) can also be used for diagnosis of bacterialvaginosis. Likewise, the pathogen Trypanosoma cruzi, which causesChaga's disease, possesses trans-sialidase (sialic acid transferase),which can also use the N-acetylneuraminic acid-firefly luciferinsubstrate. Hence, the N-acetylneuraminic acid-firefly luciferinsubstrate can also be used for detection of active infection of thispathogen. Other substrates and their applications include abeta-galactosaminidase substrate (e.g., fireflyluciferin-N-acetyl-beta-D-galactosaminide) for detection ofbeta-galactosaminidase, a L-proline aminopeptidase substrate (e.g.,L-prolyl-6-amino firefly luciferin) for detection of L-prolineaminopeptidase, a leukocyte esterase substrate (e.g., carboxylicacid-firefly luciferin or alcohol-firefly luciferin conjugates) fordetection of leukocyte esterase, an alpha-L-Fucosidase substrate (e.g.,alpha-L-fucopyranoside-firefly luciferin conjugate) for detection ofalpha-L-Fucosidase, a lipid esterase substrate (e.g., Octanoate-fireflyluciferin conjugate) for detection of lipid esterase, and ahydroxyproline aminopeptidase substrate (e.g., L-hydroxylprolyl-6-aminofirefly luciferin) for detection of L-Hydroxyproline aminopeptidase.

Furthermore, the present invention provides a method for reducing thebackground resulted from the contaminating firefly luciferin in theconjugate. The substrates disclosed in the present invention all containfirefly luciferin moiety in the substrates, which is cleaved by anenzyme to free the firefly luciferin in the conjugate. The freedluciferin enables the firefly luciferase-catalyzed biochemiluminescencereaction, hence the detection of the enzyme. The substrates aresynthesized by coupling a specific moiety to firefly luciferin, which isfollowed by purification to remove unincorporated firefly luciferin andother contaminants. However, the presence of even minute amounts ofcontaminating free firefly luciferin, e.g., 1 ng per reaction, can stillgenerate significant background. The inventors discovered that a reagentmix containing all necessary reagents and conditions suitable for boththe specific enzyme reaction (except for the enzyme itself, which may bepresent in a sample for detection) and firefly luciferase reaction(except for luciferin) could be prepared in a single mix (the mastermix). The master mix can be simply incubated for an appropriate periodof time, e.g., overnight at 2-8° C. before lyophilization or freezing orbeing used for detection, which allows the removal of contaminatingluciferin by the firefly luciferase in the master mix.

Coelenterazine and its corresponding enzyme (e.g., apoaequorin andRenilla luciferase) can be used instead of firefly luciferin and fireflyluciferase. Coelenterazine is found in radiolarians, ctenophores,cnidarians, squid, copepods, chaetognaths, fish and shrimp.Coelenterazine is the light-emitting molecule in the protein aequorin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram depicting the chemical structure ofN-acetylneuraminic acid.

FIG. 2 is a diagram depicting the chemical structure of fireflyluciferin.

FIG. 3 is a diagram depicting the chemical structure of anN-acetylneuraminic acid-firefly luciferin conjugate.

FIG. 4 is a diagram depicting the chemical structure of 4,7 methylatedN-acetylneuraminic acid-firefly luciferin conjugate.

FIG. 5 provides a schematic drawing showing the principle for influenzaneuraminidase detection as a means for the detection of influenza virususing the N-acetylneuraminic acid-firefly luciferin conjugate.

FIG. 6 is a graph showing chemiluminescence as a function of flu virusinput in the detection of the influenza virus using theN-acetylneuraminic acid-firefly luciferin conjugate.

FIG. 7 is a graph showing the reaction kinetics in a real time assay,which both the neuraminidase and luciferase reaction occursimultaneously in the same detection tube.

FIG. 8 is a diagram depicting the chemical structure of aL-proply-6-amino firefly luciferin conjugate.

FIG. 9 is a diagram depicting the chemical structures of acarboxylate-firefly luciferin ester conjugate and an alcohol-fireflyluciferin ester for detection of leukocyte esterase.

FIG. 10 is a diagram depicting the chemical structure ofalpha-L-fucopyranoside firefly luciferin conjugate for detection ofalpha-L-fucosidase.

FIG. 11 is a diagram depicting the chemical structure ofL-glycyl-L-prolyl-6 amino firefly luciferin conjugate for detection ofglycylproline dipeptidyl aminopepdidase (GPDA)

FIG. 12 is a diagram depicting the chemical structure of fireflyluciferin-N-acetyl-beta-D-galactosaminide conjugate for detection ofBeta-D-galactosaminidase.

FIG. 13 is a diagram depicting the chemical structure of a fattyacid-firefly luciferin ester conjugate for detection of Salmonella lipidesterase.

FIG. 14 is a diagram depicting the chemical structure ofhydroxyproline-firefly luciferin conjugate for detection ofhydroxyproline aminopeptidase.

FIG. 15 is a diagram depicting the chemical structure of coelenterazine.

FIG. 16 is a diagram depicting the chemical structures of the conjugatesbetween N-acetylneuraminic acid and coelenterzine or its derivatives.

Table 1: Detection Results of a Bacterial Vaginosis Test PositiveControl Using the Detection Mix

DETAILED DESCRIPTION OF THE INVENTIONS

The present invention relates to reagents and methods for neuraminidaseactivity and influenza virus detection. These reagents and methodsdisclosed in the present invention enable simple, rapid, specific andsensitive detection of influenza virus types A and B.

The present invention utilizes a group of chemicals that enable thedetection of influenza virus by detecting the neuraminidase activity ofinfluenza virus. These chemicals are conjugates betweenN-acetylneuraminic acid (sialic acid) or its derivatives and fireflyluciferin. Chemical structures of N-acetylneuraminic acid (sialic acid)and firefly luciferin are depicted in FIGS. 1 and 2.

In the current invention, the term chemiluminescence and bioluminescenceare used interchangeably, Firefly luciferin is oxidized in the presenceof the corresponding luciferase and other co-factors (e.g., ions andATP) to produce light.

One important aspect of the current invention is that theN-acetylneuraminic acid-firefly luciferin conjugate (herein frequentlyreferred to as the conjugate) is not necessarily specific for influenzaviral neuraminidase. This is because the capture reagent can selectivelybind to influenza virus, thereby resulting in specific detection ofinfluenza virus even if the conjugate is not specific for influenzavirus. More importantly, this conjugate is a more natural and,potentially more efficient, substrate for neuraminidase as compared tothe methylated substrate.

One preferred embodiment of the N-acetylneuraminic acid-fireflyluciferin conjugate is depicted in FIG. 3. The conjugate is linkedtogether via a glycoside bond through the 2′ position ofN-acetylneuraminic acid and —OH group on the aromatic ring of fireflyluciferin. It is understood that variations of such linkage may beappropriate for neuraminidase activity detection. Similarly, derivativesof N-acetylneuraminic acid and firefly luciferin may also be appropriatefor neuraminidase activity detection described in this invention. Theconjugates used as neuraminidase substrate can be represented by thefollowing formula shown for the Na salt:

wherein R1, R2, R3 are each, independently of one another, hydrogen oralkyl having 1-5 carbon atoms.

A number of organisms express neuraminidase that may hydrolyze the sameN-acetylneuraminic acid-firefly luciferin conjugate described above. Forexample, parainfluenza and certain bacterial species possessneuraminidase. Thus, this class of substrate may not be specific forneuraminidase from a particular organism. Therefore, the reagents andmethods described in the current invention can also be used fordetection of other sialidases of bacterial, viral, protozoa, andvertebrate (including human) origin besides sialidase from influenzavirus.

Sialic acid derivatives are also appropriate for use in influenza virusdetection provided that the derivatives can form a glycoside withfirefly luciferin or its derivatives, and that the conjugate can becleaved by neuraminidase. Examples of sialic acid derivatives include4-alkyl or 7-alkyl or 4,7 alkyl N-acetylneuraminic acids (e.g. thosedescribed in U.S. Pat. No. 6,303,764 and U.S. Pat. Nos. 6,420,552,6,680,054). One example (4,7 methylated N-acetylneuraminic acids-fireflyluciferin conjugate) is given below in FIG. 4, which has betterspecificity for flu neuraminidase than for bacterial neuraminidase.

Methods for synthesizing the conjugate depicted in FIG. 3, or similarconjugates, can be derived from existing methods. For example, a methodfor synthesizing N-acetylneuraminic acid-dioxetane conjugate, describedin a publication (e.g., Analytical Chemistry 280, 291-300), can be usedfor synthesis of the conjugate depicted in FIG. 3, or similarconjugates. An example is included in EXAMPLE 1.

The principle for detecting neuraminidase activity using the conjugateis depicted in FIG. 5. The sialic acid-firefly luciferin conjugate is asubstrate for neuraminidase, which cleaves the substrate to give rise tofree firefly luciferin that is the substrate of luciferase, which ispresent in the detection mix. The conjugate itself is not a substratefor firefly luciferase. Therefore, the firefly luciferase-catalyzedbiochemiluminescence is dependent on neuraminidase activity, which isprovided by the influenza virus or its lysate in a sample.

In an influenza virus detection assay using a sialic acid-luciferinconjugate, the sample containing influenza virus is first incubated withthe capture reagent. After binding of influenza virus to the reagent,the reagent is washed with wash buffer. The capture reagent, now boundwith influenza virus, is suspended with the conjugate mix and thenincubated for an optimal period of time and at optimal temperature toallow neuraminidase-dependent cleavage of the conjugate. The capturereagent is then removed using an appropriate means. The supernate isthen added to the detection mix, which contains luciferase and othernecessary chemicals, and placed in a luminometer to detect the lightsignal. A more detailed protocol is provided in EXAMPLE 1.

In some embodiments, detection of influenza virus using the conjugateinvolves essentially three steps: 1) capture of influenza virus usingthe capture reagent, 2) incubation of the conjugate with the capturedvirus or its lysate, and 3) detection of the cleaved conjugate bydetecting the resulting luciferin using luciferase, which generates thelight signal in the presence of luciferin and other appropriatereagents. In certain embodiment, steps 2 and 3 can be combined togetheras a single step thereby reducing the number of procedure intoessentially two. More detailed description of the detection proceduresare disclosed in Example 1 and Example 2. An example of the test resultis provided in FIG. 6.

In other embodiments, detection of influenza virus using the conjugateinvolves essentially 2 steps: 1) incubation of the conjugate with thecaptured virus or its lysate, and 2) detection of the cleaved conjugateby detecting the resulting luciferin using luciferase, which generatesthe light signal in the presence of luciferin and other appropriatereagents. In certain embodiment, step 1 is eliminated through the use ofspecific conjugates for influenza viral neuraminidase or inhibitors fornonspecific neuraminidase activities, thereby reducing the number ofprocedure into essentially one (the step 2).

In one embodiment, the method for detecting influenza virus in a samplecomprises essentially three steps: a) virus capture, b)neuraminidase-dependent cleavage of the conjugate to release fireflyluciferin moiety, which becomes free firefly luciferin in the presenceof neuraminidase in a sample, and c) firefly luciferase-catalyzedbiochemiluminescence reaction to detect the released luciferin. The keyfeature of his method is the separation of neuraminidase reaction fromthe biochemiluminescence reaction. The corresponding test kit containsthree key components (the capture reagent, conjugate mix, and detectionmix) and two accessory components (wash buffer and conjugate suspensionsolution or deionized water). The capture reagent can be a solid supportsuch as magnetic particles, which are coated with influenza virusspecific receptors, e.g., monoclonal antibodies against the surfaceprotein hemagglutinin. Examples of compositions for these components aredescribed in EXAMPLE 1. A detection protocol for using this test kittype is described in EXAMPLE 1.

Yet in another embodiment, the method for detecting influenza virus in asample comprises essentially two steps: a) virus capture, and b) fireflyluciferase-catalyzed biochemiluminescence reaction to detect freeluciferin as soon as luciferin moiety is cleaved from the conjugate byinfluenza neuraminidase. The key feature of this method is thecombination of neuraminidase reaction with the biochemiluminescencereaction in a single step. This method is therefore referred to as realtime detection of influenza neuraminidase or real time detection methodsince the free luciferin cleaved from the conjugate is immediatelyconsumed by luciferase to give rise to light signal. The test kit usedin this detection method contains two key components (the capturereagent and detection mix) and two accessory components (the wash bufferand virus lysis solution). The capture reagent can be the same as thatused in the three step procedure.

In the real time neuraminidase detection method, the detection mixcontains all necessary chemicals and appropriate buffer forneuraminidase reaction, including the conjugate itself, and for fireflyluciferase-catalyzed biochemiluminescence reaction except for fireflyluciferin. Examples of the composition for these components aredescribed in EXAMPLE 2. Detection of influenza virus in a sample usingthis version of the test kit, or variations of it, consists ofessentially two steps: 1) capture of influenza virus in a samplefollowed by washing and, 2) detection of the captured virus or itslysate using the detection mix. The optimal pH for neuraminidasereaction and firefly luciferase-catalyzed biochemiluminescence, i.e., pH6.5 vs. pH 7.8, are different. It is surprising that both enzymes havesufficient activity at a mutual pH, e.g., at or around 7, which can beused for the detection mix for both reactions. The combination of thetwo reactions considerably simplifies the detection procedure. A moredetailed detection protocol is described in EXAMPLE 2.

Yet still in another embodiment, the virus capture step is eliminatedfrom the detection procedure, thereby enabling essentially a one-stepdetection procedure. Specific detection of influenza virus is enabledthrough the use of one of two approaches or a combination of both. Incertain aspects of the current invention, specific detection ofneuraminidase from a particular source is achieved using a modifiedsialic acid moiety of the N-neuraminic acid-firefly luciferin conjugate.Examples of conjugates with modified sialic acid derivatives, e.g.,4-alkyl or 7-alkyl or 4,7 alkyl N-acetylneuraminic acids are specificfor influenza viral neuraminidase over bacterial neuraminidase asdescribed in U.S. Pat. Nos. 6,303,764, 6,420,552, and 6,680,054, whichare cited here solely for reference. An example of such a conjugate isdepicted in FIG. 4.

In another embodiment, the undesired neuraminidase is inhibited usingspecific antibodies and inhibitors. The undesired neuraminidase activityis inhibited using specific polyclonal or monoclonal antibodies. Forexample, for detection of influenza viral neuraminidase, thenon-specific neuraminidase activity from likely contaminating organismsin the sample such as bacterial species Streptococcus pneumoniae andActinomyces viscosus are inhibited using antibodies specific for theneuraminidases from these sources. This approach is possible becauseneuraminidases from different organisms have distinct amino acidsequences, which permits the generation of species-specific, orsub-species-specific, neuraminidase antibodies. For example, specificantibodies are commonly used to differentiate neuraminidase subtypes ofinfluenza virus in neuraminidase neutralization assays.

The procedure of making these antibodies is well to the skilled in theart. For example, recombinant neuraminidase protein can be produced inE. coli by cloning the complete or partial genomic (in the case ofbacterial neuraminidase) or cDNA (in the case of eukaryoticneuraminidase) sequences into a bacterial expression vector thatpreferably contains an affinity ligand, e.g., his-tag, which facilitatesthe purification of the recombinant protein. Bacterial clones expressingthe enzyme can be screened and selected in a chemiluminescence assayusing the N-neuraminic acid-firefly luciferin conjugate. If therecombinant enzyme is tagged with an affinity ligand, then appropriateaffinity column can be used to purify the enzyme. For example, nickelcoated agarose column can be used to purity his-tagged recombinantneuraminidase.

The purified neuraminidase can be used to immunize an animal, e.g., arabbit, for production of polyclonal antibodies. Alternatively, theprotein can be used to immunize mouse from which the B-lymphocytes canbe used to generate hybridoma, which can be used for screening amonoclonal antibody that specifically inhibits the neuraminidase.

One or more monoclonal and/or polyclonal antibodies can be used in anassay for inhibiting neuraminidase activities from one or morecontaminating sources. For example, polyclonal antibodies or anti-seraor monoclonal antibodies for Streptococcus pneumoniae, Actinomycesviscosus and parainfluenza neuraminidases can be used in an assay fordetecting influenza viral neuraminidase. It is understood that theamounts of each antibody or anti-serum used in an assay need to beoptimized so that the antibodies can maximally inhibit contaminatingneuraminidase activities but not the neuraminidase activity to bedetected. Likewise, small molecules specific for the undesiredneuraminidases, e.g., bacterial neuraminidase, can also be used infashion similar to the antibodies.

Yet another embodiment involves the use of the neuraminic acid-fireflyluciferin depicted in FIG. 3 for specific for influenza virus infectionusing an influenza viral neuraminidase specific inhibitor such asoseltamivir carboxylate. To perform specific detection of influenzavirus in a sample, two forms of the detection mix disclosed in EXAMPLE 2need to be formulated. One form (Detection Mix 1) contains no influenzaneuraminidase inhibitor and the other (Detection Mix 2) contains theinhibitor. For clinical diagnosis, a clinical sample such as nasal swabwash is split into two portions. One portion of the sample is added toDetection Mix 1 whereas the other portion is added to Detection Mix 2,followed by detection of lights signal as described in EXAMPLE 2.

If both are positive, it indicates that the neuraminidase activity inthe sample is from a non-influenza virus source, such as bacteria orparainfluenza virus. If Detection Mix 1 is positive and Detection Mix 2is negative or has significantly reduced signal, then it indicates thatthere is influenza virus infection. If both are negative, then itindicates that there is no infection with influenza virus orparainfluenza virus. Appropriate influenza viral neuraminidaseinhibitors include, but are not limited to, oseltamivir carboxylate,zanamivir, and peramivir.

[List Some and Give References Disclosing Such Inhibitors.]

A preferred embodiment is the real time detection method, which combinesthe neuraminidase reaction, i.e., cleavage of the conjugate, and fireflyluciferase-catalyzed biochemiluminescence reaction together in a singlereagent mix. The light signal is measured at real time, i.e., fireflyluciferin is detected as soon as it is cleaved from the conjugate byneuraminidase. In this real time detection method, there is noaccumulation of free luciferin. Since both the neuraminidase and fireflyluciferase catalyzed reactions occur in the same reaction mix, the pHcan not be exactly optimal for both neuraminidase and fireflyluciferase. This is in contrast with the three step method, where thetwo reactions are separated, which allows the two reactions to occur atoptimal pH (around pH 6.5 for neuraminidase and 7.8 for fireflyluciferase) and the released firefly luciferin to accumulate for anextended period of time (e.g., 10 minutes) before being subjected tofirefly luciferase-catalyzed biochemiluminescence reaction. Therefore,the signal output with the real time detection method (e.g., 10 seconds)may be lower.

Lower signal output in the real time method can be partially compensatedby extending the signal collection time from, for example, 10 seconds to120 seconds. In addition, lower activity of the firefly luciferase at pH7.0, which is lower than the optimal pH (pH 7.8), can be partially orcompletely compensated by adding more luciferase to the reaction mix.Moreover, the background counts is expected to be lower in the real timemethod since auto-cleavage (i.e., neuraminidase-independent) of theconjugate at lower pH is more severe. Consequently, the signal tobackground ratio for the real time method is expected to be similar to,or no less than, that for non-real time detection method. Surprisingly,the real time assay as described in EXAMPLE 3 gave excellent sensitivityfor the detection of influenza virus.

The real time method offers a number of advantages, including 1) moreconvenience due to the combination of two reactions in one test tube, 2)better specificity due to real time measurement of the neuraminidaseactivity, 3) less background because of less auto-cleavage of theconjugate at neutral pH, 4) shorter operation time and therefore fastertime-to-result due to the combination of the two reactions into one, 5)more quantitative, and 6) lower cost of manufacturing because there isonly one key reagent mix. Thus, the real time detection method is apreferred choice for the detection of influenza virus in many situationssuch as point-of-care use.

It is understood that influenza virus capture step is not necessary forcertain applications. For monitoring influenza virus growth in aculture, for example, the medium can be diluted in an appropriate bufferor water and directly added to the conjugate mix according to the threestep protocol described in example 1 or to the detection mix accordingto the real time protocol described in example 2. Example 3 describesthe detection of cultured virus in a solution without virus capturestep. When the influenza neuraminidase specific substrate (e.g. theconjugate described in FIG. 4) is used, capture step is not necessary.

It is further understood that the reagents and methods disclosed aboveare also applicable for the detection of other diseases or pathogens.For example, elevated sialidase activity in vaginal fluid samples isindicative of bacterial infection in vagina, which is known as BacterialVaginosis (BV). Thus, an embodiment of the current invention is the useof the neuraminic acid-firefly luciferin conjugate depicted in FIG. 3for diagnosis of BV, which is described in more detail in EXAMPLE 4.

Another embodiment is the use of the neuraminic acid-firefly luciferinconjugate depicted in FIG. 3 for diagnosis of active infection by theparasite Trypanosoma cruzi, which causes Chagas' disease. Trypanosomacruzi expresses high levels of trans-sialidase activity, which is a typeof sialidase. The neuraminic acid-firefly luciferin conjugate depictedin FIG. 3 can be used to measure trans-sialidase activity and thus usedto diagnosis of active infection of this parasite. This application isdescribed in more detail in EXAMPLE 6.

Other applications include a L-proline aminopeptidase assay usingL-prolryl-6-amino firefly luciferin as the substrate for detection ofthe enzyme (EXAMPLE 7), a leukocyte esterase assay using fireflyluciferin-alcohol or firefly luciferin-carboxylate esters as substratefor detection of esterase activity (EXAMPLE 8), an alpha-L-fucosidaseassay using an alpha-L-fucopyranoside-firefly luciferin conjugate as thesubstrate for detection of alpha-L-fucosidase (EXAMPLE 9), aglycylproline dipeptidyl aminopeptidase assay usingL-glycyl-L-prolyl-firefly luciferin conjugate as the substrate fordetection of the enzyme (EXAMPLE 10), a beta-D-galactosaminidase assayusing firefly luciferin-N-acetyl-beta-D-galactosaminide as the substratefor detection of beta-galactosaminidase (EXAMPLE 11), a lipid esteraseassay using octanoate-firefly luciferin ester for detection of a lipidesterase (EXAMPLE 12), and L-hydroxylprolyl-6-amino luciferin conjugatefor detection of L-Hydroxyproline amiopeptidase (EXAMPLE 13).

Within the scope of the present invention is a manufacturing processthat results in greatly reduced background light signal. The basicdetection principle disclosed in this invention involves the use offirefly luciferase in a chemiluminescence reaction to detect freefirefly luciferin, which is cleaved from a firefly luciferin conjugateby a marker enzyme, e.g., neuraminidase. The luciferin conjugate isnormally synthesized by using a large quantity of luciferin. Normally,there is still significant amounts of free luciferin, e.g., up to 90%,which remains free after conjugation reaction and has not beenincorporated into the conjugate. This free firefly luciferin remains asignificant contaminant that contributes to high background even aftercareful HPLC purification. This is because even if the free fireflyluciferin accounts for only 0.1% in the purified conjugate, there isstill 0.1 ng of free firefly luciferin if 1 microgram of conjugate isused in a reaction. The presence of even 0.1 ng of free fireflyluciferin can generate significant background signal.

The inventors discovered that the background signal from thecontaminating free firefly luciferin gradually decreases over a periodof several hours even at refrigerated conditions in the master mix forreal time detection of neuraminidase, which contains reagents for boththe neuraminidase and firefly luciferase reactions. This is because thecontaminating free firefly luciferin is consumed by the fireflyluciferase. Thus, the use of a reagent mix for real time neuraminidasedetection provides another advantage in that it allows the removal ofcontaminating free firefly luciferin with a simple method. A preferredembodiment involves the incubation of the reagent mix for real timedetection for an appropriate period of time at room temperature or atrefrigerated temperature until the background signal reaches acceptablelevel before being lyophilized or frozen during manufacturing. Theincubation time is at least 2 hours, at least 4 hours, at least 6 hours,or at least 8 hours. The most convenient and thus preferred incubationcondition is an overnight incubation at 2-8° C. or refrigeratedtemperature. It is understood that the amounts of firefly luciferase andother components in the reagent mix need to be optimized to account forthe loss of activity during the incubation. An example of the reagentmix for real time detection and its manufacturing are described inEXAMPLE 2.

Coelenterazine and its conjugates are depicted in FIG. 15 and FIG. 16.

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The following preferred specific embodiments are,therefore, to be construed as merely illustrative, and not limitative ofthe remainder of the disclosure in any way whatsoever.

In the foregoing and in the following examples, all temperatures are setforth uncorrected in degrees Celsius and, all parts and percentages areby weight, unless otherwise indicated.

EXAMPLE 1 Test Kit Formulation and Influenza Virus Detection

Synthesis of N-acetylneuraminic Acid-firefly Luciferin Conjugate

Methods for synthesizing the conjugate depicted in FIG. 3, or similarconjugates, can be derived from existing methods. For example, a methodfor synthesizing N-acetylneuraminic acid-dioxetane conjugate, describedin a publication (e.g., Analytical Biochemistry 280, 291-300), can beused for synthesis of the conjugate depicted in FIG. 3, or similarconjugates. An example for the synthesis of the N-acetylneuraminicacid-firefly luciferin conjugate is described as follows:Methyl(5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-b-D-alactononulopyranosylchloride) Onate was prepared in two steps from a commercially availableN-acetylneuraminic acid as described a publication (AnalyticalBiochemistry 280, 291-300), which is cited here solely for reference.The crude chloride was purified by a silica gel plug, and eluted with200 mL of 80-90% EtOAc in hexanes. After concentration of the filtrate,the chloride was obtained as a white powder and immediately used for thenext coupling reaction.

Luciferin (0.83 g) and the phase transfer catalyst tetrabutylammoniumhydrogen sulfate (2.43 mmoles) was placed in a 100-mL round-bottomedflask and treated with 12.5 mL of CH₂Cl₂ and 17.5 mL of 0.5N NaOH atroom temperature, which forms a two phase mixture. 1.24 grams of thechloride prepared as described above was dissolved in 5 mL of CH₂Cl₂ andadded to the mixture. After an hour of vigorous stirring, the reactionmixture will be diluted with CH₂Cl₂ and loaded into a separator funnelthat contains saturated sodium bicarbonate solution. After the organiclayer is separated, the aqueous layer is further extracted twice withCH₂Cl₂. The combined organic layers was washed with H₂O and dried overanhydrous Na₂SO₄. The organic solution was treated with 10 drops of Et₃Nand concentrated. The crude product was purified by the silica gelchromatography.

The resulting pyranoside was deprotected at 0° C. for 5 minutes in amixture of 6.5 mL of THF, 6.5 mL of MeOH and 12 mL of 1 N NaOH. Themixture was further stirred at room temperature for an hour, followed byaddition of 1.05 g solid sodium bicarbonate to lower the pH. Thesolution was filtered using a Buchner funnel, rinsed with a small volumeof water, and pooled together for reverse-phase preparative HPLCpurification.

Purification of the Conjugate

The peak identity and profile were first established using ananalytical/semi-preparative Mass spectrometry-HPLC (MASS-HPLC) and acolumn similar to a C-18 column. The peak corresponding to the expectedmolecular weight was collected and used in an assay to detect bacterialneuraminidase using a two step assay, which is similar to what isdescribed below. This peak was subsequently used as a “standard” toguide preparative HPLC purification.

A preparative HPLC with C-18 column was used for relatively large scalesubstrate purification using an isogradient mobile phase solution (1%ammonium acetate, pH 7.0: methanol=20%: 80%). The expected peak wascollected and confirmed with an analytical HPLC by comparing theretention time of the standard. The peak was collected and pooled frommultiple runs. Based on analytical HPLC, the purity of the substrate wasmore than 98%. The substrate concentration was estimatedspectrophotometrically using luciferin as the standards (absorbancewavelength 336 nm).

Use of the Conjugate for Influenza Virus Detection

The protocol described here is a two-step assay, i.e., the neuraminidasereaction and firefly luciferase reaction are carried out sequentially intwo steps. Approximately 1 μg of conjugate was incubated at 37° C. for15 minutes with 0, 0.5, 5, 50, 500 or 5,000 TCID₅₀ units of influenzavirus (A/WS/33) in a 25 μL reaction solution (32.5 mM MES, pH 6.5, 4 mMCaCl₂, 0.65% Triton X-100). 12.5 μL of the reaction solution was mixedwith 100 μL of luciferase reaction solution (20 nM recombinant fireflyluciferase, 4 mM ATP, 13.4 mM MgSO₄, 100 mM Trizma, pH 7.9, 4% mannitol,and 1% sucrose) and measured for light signal using a luminometer(Sirius purchased from Berthold). Total light output was collected byintegrating RLU over a period of 30 seconds.

As shown in FIG. 6, 50 TCID₅₀ units of influenza virus could result insignificantly more signal in comparison with the negative control. Inaddition, linear response of the signal was also observed over theentire range that was tested.

An Influenza Virus Detection Kit

In this example, the influenza test kit contains three key components:the capture reagent, conjugate mix and detection mix. In addition to thekey components, there are two accessory components: deionized water andwash buffer. Examples of the compositions of these components are listedbelow:

Capture Reagent

-   -   10 mg/mL influenza virus specific antibody (polyclonal or        monoclonal) coated magnetic particles in 0.1% sodium azide

Conjugate Mix (Lyophilization is Preferred)

MES, pH 6.5 32.5 mM CaCl₂ 4 mM BSA 1 miligram/mL Triton X-100 0.5%  Mannitol 4% Sucrose 1% Conjugate 10 microgram/mL

Detection Mix (Lyophilization is Preferred)

Trizma, pH 7.8 100 mM MgSO₄ 15 mM BSA 1 mg/mL ATP, Na Salt 12 mM NP₄₀ orEquivalent 0.1%   DTT 10 mM Co-enzyme A 1 mM EDTA, Na Salt 2 mM Mannitol4% Sucrose 1% Firefly Luciferase 2-4 micrograms/mL

Wash Buffer

-   -   0.5× PBS

Deionized Water

-   -   Deionized water is used to suspend the conjugate mix and        detection mix if these components are in a lyophilized form.

Detection Protocol

The influenza virus detection assay comprises essentially threesteps: 1) influenza virus capture, 2) cleavage of sialic acid-fireflyluciferin conjugate with influenza neuraminidase, and 3) detection ofreleased firefly luciferin. Specifically one can use the following basicprotocol:

-   -   1. Add 100 μL of capture particles (1 mg of capture reagent) to        1.0 mL sample. In this example, the capture reagent is magnetic        particles coated with an influenza virus specific antibody.    -   2. Incubate at room temperature for 5-10 minutes.    -   3. Wash the reagent twice with 1 mL wash buffer using a magnet.    -   4. Suspend the washed capture reagent, now bound with flu virus        if there is influenza virus in the sample, in 100 μL conjugate        solution, which will lyse the virus because of the presence of        Triton X-100.    -   5. Incubate at room temperature for 10-15 minutes.    -   6. Using a magnet, recover and transfer the supernate (100 μL)        to 100 μL firefly luciferase reaction solution pre-loaded into a        detection tube, or a lyophilized detection mix in a detection        tube.    -   7. Place the detection tube into a luminometer and record the        light signal (relative light unit) for an appropriate period of        time (e.g., 30 seconds).

Note that firefly luciferase mediated biochemiluminescence reaction isof a glow light type, which stably emits light for at least 5 minutes.Therefore, there is no need to use a luminometer with an automatedinjector.

EXAMPLE 2 Real Time Detection of Influenza Viral Neuraminidase using theNeuraminic Acid-Firefly Luciferin Conjugate for the Detection ofInfluenza Virus

In this example, influenza viral neuraminidase activity is detected atreal time, i.e., neuraminidase reaction and biochemiluminescencereaction are combined in a single reaction tube and carried outsimultaneously. A master mix that contains all necessary componentsexcept for neuraminidase and free firefly luciferin was formulatedaccording to the composition for the Detection Mix (see below). Afteradjusting the pH to 7.0 to 7.2 and the final volume, the mix is storedat 2-8° C. overnight, which allows the luciferase consume thecontaminating free luciferin and thus reduces the background. Thedetection mix is then aliquoted into vials (1.0 mL each), lyophilized,and stored at −20° C.

For influenza virus detection, one vial of the lyophilized detection mixis suspended in 0.9 mL of buffer containing 1% Triton X 100 or NP-40equivalent, and 5 μL/mL Antifoam A (Sigma). Prior to detection, 90 μL ofthe reconstituted detection mix was transferred to a detection tube. 10μL of sample was added to the detection mix, followed by luminometerreading for a period of 60 minutes to document the reaction kinetics.

FIG. 7 depicts the reaction kinetics of an assay that was performedusing a protocol that is substantially similar to that described above.As shown, the signal intensity increased rapidly in the first 10 minutesin the presence of influenza virus, followed by a period of plateau thatlasted up to at least 60 minutes. The reactions reached plateau at 10minutes, after which the light signal intensity remained fairlyconstant. For all reactions with input influenza virus, little or nodecrease in signal intensity was observed even at 60 minutes (FIG. 7).Thus, test results obtained at any time point between 10 and 60 minutesare likely to be valid.

An Influenza Virus Detection Kit

According to this example, the influenza test kit for this assay formatcontains two key components: the capture reagent and detection mix, thelatter containing both the conjugate and all necessary reagents forfirefly luciferase catalyzed biochemiluminescence except for fireflyluciferin. In addition to the key components, there are two accessorycomponents: Virus Lysis Buffer and Wash Buffer. Examples of thecompositions of these components are listed below:

Capture Reagent

-   -   10 mg/mL influenza virus specific antibody coated magnetic        particles in 0.1% sodium azide

Detection Mix (Lyophilized Form is Preferred)

Imidazole, pH 7.0 50 mM BSA 1 mg/mL ATP, Na Salt 12 mM DTT 10 mMCo-enzyme A 1 mM MgSO₄ 15 mM CaCl₂ 4 mM Mannitol 4% Sucrose 1% Conjugate10 μg/mL Firefly Luciferase 4-5 μg/mL (thermal stable firefly luciferaseis preferred. Thermal stable firefly luciferase is available fromcommercial vendors such as Promega.).

Wash Buffer

-   -   0.5× PBS

Virus Lysis Buffer

Imidazole, pH 7.0 10 mM Triton X-100 0.5%

Preparation of the Detection Mix

The detection mix is prepared by mixing stock solutions of the reagentslisted above with deionized water (approximately 70%). The pH of the mixsolution is adjusted with 1 N NaOH or KOH to between 7.0 and 7.2. Thesolution is then adjusted to the final volume with deionized water,stored overnight at 2-8° C., aliquoted 0.5 mL (or other desired volume)into appropriate lyophilization vials, lyophilized, and stored atappropriate temperature (e.g., at or below -20° C. for long termstorage).

Detection Protocol

The influenza virus detection assay comprises essentially two steps: 1)influenza virus capture and 2) cleavage of neuraminic acid-fireflyluciferin conjugate with influenza neuraminidase and simultaneousdetection of the released firefly luciferin. Specifically one can usethe following basic protocol:

-   -   1. Bring the lyophilized detection mix reagent to room        temperature.    -   2. Add 100 μL of capture particles (1 mg of capture reagent) to        1.0 mL sample. In this example, the capture reagent is magnetic        particles coated with influenza specific antibody.    -   3, Incubate at room temperature for 5-10 minutes.    -   4. Wash the reagent twice with 1 mL wash buffer using a magnet.    -   5. Suspend the washed capture reagent, now bound with influenza        virus if there is influenza virus in the sample, in 500 μL virus        lysis solution, which lyses the virus because of the presence of        Triton X-100.    -   6. Using a magnet, recover and transfer the supernate (500 μL)        to the lyophilized detection mix. Mix, transfer the solution to        a detection tube, and incubate at room temperature for 5-10        minutes.    -   7. Place the detection tube into a luminometer and record the        light signal (relative light unit) for an appropriate period of        time (e.g., 120 seconds).

Note that the detection mix can be suspended in 0.25 mL of lysis bufferto make 2× solution. Smaller amounts such as 25 microliters can be usedto mix 25 microliters of samples such as culture influenza virus fordetection. The influenza virus capture step can be eliminated if aspecific substrate, such as the one that is depicted in FIG. 4, is used.In this case, the assay procedure becomes essentially one step.

EXAMPLE 3 Use of Antibodies to Inhibit Non-Specific NeuraminidaseActivity in an Influenza Viral Neuraminidase Assay

The commonly used clinical samples for influenza detection are throatand nasal swabs. Some bacterial species that are found in nasal or oralcan also secret neuraminidase. These bacterial species includeStreptococcus pneumoniae and Actinomyces viscosus. In this example,monoclonal or polyclonal antibodies specific for the neuraminidases forthese bacterial species are added to the conjugate mix in Example 1 orthe lysis buffer or detection mix in Example 2. Bacterial neuraminidasein the sample, if any, is blocked by the antibodies thereby reducing thebackground. The optimal amounts of antibodies need to be determinedexperimentally.

EXAMPLE 4 Diagnosis of Influenza Infection using an N-acetylneuraminicAcid-Luciferin as the Chemiluminescent Substrate and an Influenza ViralNeuraminidase Inhibitor

As discussed, the neuraminic acid-firefly luciferin depicted in FIG. 3is not specific for a particularly source of neuraminidase. However,highly specific inhibitors for influenza virus neuraminidase such asoseltamivir are available. To perform specific detection of influenzavirus in a sample, two forms of the detection mix disclosed in EXAMPLE 2need to be formulated. One form contains no influenza neuraminidaseinhibitor (Detection Mix 1) and the other contains the inhibitor(Detection Mix 2). A clinical sample such as nasal swab is split intotwo portions. In a parallel detection, one portion of the sample ismixed with Detection Mix 1 whereas the other portion is mixed withDetection Mix 2, followed by detection of lights signal as described inEXAMPLE2.

If both are positive, it indicates that either the sample iscontaminated with other bacterial neuraminidase or there is other viralinfection such as infection with parainfluenza virus, which also hasneuraminidase activity. If Detection Mix 1 is positive while DetectionMix 2 is negative, then it indicates that there is influenza virusinfection. If both are negative, then it indicates that there is noinfection with influenza virus or parainfluenza virus.

EXAMPLE 5 Diagnosis of Bacterial Vaginosis using an N-acetylneuraminicAcid-Luciferin as the Chemiluminescent Substrate

In this example, an N-acetylneuraminic acid-firefly luciferin conjugateis used as the chemiluminescent substrate for detection of bacterialvaginosis. A detection mix similar to that described in Example 2 can beused for this purpose. However, it is understood that the detection mixmay need to be optimized for bacterial vaginosis detection sincesialidase activity in a vaginal sample may be considerably higher thanthat for a sample for influenza detection.

The detection mix solution described in Example 2 is used to demonstratethe detection of bacterial vaginosis. The Osom BV blue positive controland negative control (from Genezyme Diagnostics), which containdifferent levels of bacterial sialidase, are used to demonstrate the useof the detection mix for bacterial vaginosis detection. 40 microlitersof the control sample at various dilutions (1:10, 1:100, 1:1000, and1:10000) was spiked into a Hygiena Snapshot swab, which was placed in500 microliters of detection mix described in Example 2 and immediatelyplaced in a handheld luminometer (Hygiena) for detection. Fourreplicates were tested with each dilution sample and with the negativecontrol as well. Total assay time: <2 min.

For comparison, the same samples were also tested with a competing, FDAapproved product (the BV Blue test) that is based on colorimetricmethod, which is a qualitative method, involves two steps and takesabout 15 minutes. The data is presented in Table 1.

For detection that uses a patient sample, i.e., vaginal swab, thevaginal swab can be first rinsed in a sample buffer, e.g., 1 mL of 1×PBS buffer, which or a portion of which is mixed with the detection mixin solution form or, preferably, in lyophilized form in a test tube. Thetest tube is placed in a luminometer for detection. It is understoodthat a cutoff value in terms of light signal intensity (RLU) needs to beestablished by testing a large number of negative and positive samples,preferably more than 100 positive samples and 100 negative samples.

EXAMPLE 6 Diagnosis of Chagas' Disease using an N-acetylneuraminicAcid-Luciferin as the Chemiluminescent Substrate

Chagas' disease (also called American trypanosomiasis) is a humantropical parasitic disease, which occurs in the Americas, particularlyin South America. Its pathogenic agent is a flagellate protozoan namedTrypanosoma cruzi, which is transmitted to humans and other mammalsmostly by blood-sucking bugs of the subfamily Triatominae (FamilyReduviidae). The cell invasion form of T. cruzi, Trypomastigote,expresses high levels of trans-sialidase activity; therefore,measurement of sialidase level can be used for diagnosis of active T.cruzi infection and for monitoring disease or therapeutic progress.

As in the bacterial vaginosis diagnostic test, the N-acetylneuraminicacid-luciferin including their derivatives, e.g., the one depicted inFIG. 2, can be used for detection of T. cruzi infection Chaga's disease.

There are several different types of T. cruzi infection status: 1) acuteinfection, which is an acute phase of an infection, 2) chronic activeinfection, which an infection with persistent and active infection, and3) cleared or dormant infection, which is an infection without activeinfection but with detectable antibodies specific for the protozoa.

For diagnosis of acute and active or chronic infection, sialidaseactivity in plasma or serum is measured using the chemiluminescent assaydescribed in this invention. Elevated sialidase activity in plasma orserum indicates active T. cruzi infection. In an active infection assay,serum or plasma sample with appropriate dilution in a buffer (e.g., PBSbuffer) as determined with experiments is added to a detection mix asdescribed in Example 2. The detection mix is preferably lyophilized forlong-term storage. The output signal is then measured with aluminometer. Again, a cutoff value needs to be established by testing alarge number of negative samples, e.g., 100 or more of negative samples.Sensitivity of the assay can be determined by testing a large number ofpositive samples, e.g., 100 or more of positive samples confirmed withanother method such as polymerase chain reaction (PCR) method.

T. cruzi infection may be cleared by the host but still results indetectable antibodies, which can be detected with a neutralization assaythat uses the same detection mix as for an active infection test exceptthat the detection mix also contains small amounts of T. cruzisialidase. In the absence of specific antibodies in a serum or plasmasample, the T. cruzi sialidase in the detection mix generates adetectable light signal at certain level. In the presence of specificantibodies in a serum or plasma sample, a reduction of the signal can bedetected. Thus, a reduction of the signal indicates chronic, cleared ordormant infection. Again, a cutoff value needs to be established bytesting a large number of negative samples, e.g., 100 or more ofnegative samples. Sensitivity of the assay can be determined by testinga large number of positive samples, e.g., 100 or more of positivesamples confirmed with another method such as an ELISA test.

EXAMPLE 7 Chemiluminescent Proline Aminopeptidase Assay for BacterialVaginosis Diagnosis Detection

Elevated proline aminopeptidase activity in vaginal fluid has beenassociated with bacterial vaginosis. Thus, a praline aminopeptidaseassay can also be used to diagnose bacterial vaginosis. Prolineaminopeptidase (or called proline iminopeptidase) is a hydrolase thatcleaves the L-proline residues from the N-terminal position in peptides.

A substrate that can be used in a proline aminopeptidase assay forbacterial vaginosis diagnosis is L-proline-6-amino firefly luciferinconjugate (L-prolyl-6-amino firefly luciferin, FIG. 8) or itsderivatives. In a proline aminopeptidase assay, the enzyme cleaves thesynthetic substrate and releases the free 6-amino firefly luciferin,which can be quantitatively detected in a chemiluminescence reaction inthe presence of firefly luciferase and ATP. It can be a one-step assayor two step assays in a fashion similar to those described in theinfluenza or bacterial vaginosis sialidase assay (Examples 1 and 2).

The linkage between the L-proline and firefly luciferin is a peptidebond (FIG. 8). Thus, synthesis of this group of conjugates can beaccomplished using peptide synthesis chemistry, which is well known tothe skilled in the art. The substrate can be purified using HPLC andC-18 column by following a protocol similar to that described in EXAMPLE1 for the purification of N-acetylneuraminidase-firefly luciferinconjugate. Likewise, formulation of detection mix and detection protocolcan also be derived from those described in Examples 1 and 2. Forclinical diagnosis use, again a cutoff value needs to be established bytesting a large number of negative samples, e.g., 100 or more ofnegative samples. Sensitivity of the assay can be determined by testinga large number of positive samples, e.g., 100 or more of positivesamples.

EXAMPLE 8 Chemiluminescent Assays for Leukocyte Esterase Detection

Elevated leukocyte esterase (LE) activity in urinary track indicates thepresence of white blood cells and other abnormalities associated withinfection or inflammation, e.g., urinary track infection. Therefore,leukocyte esterase test (LE test) is widely used to diagnose activeurinary tract infection. LE tests are also used to screen for gonorrheainfection, colpitis, amniotic fluid infections, bacterial meningitis,and ascite or hydrothorax infection by testing leukocyte esteraseactivity in appropriate samples. Current tests generally suffer frominsufficient sensitivity and/or quantitation capability and consequentlyhave limited clinical utility.

The present invention discloses chemiluminescent substrates and methodsfor leukocyte esterase test. The appropriate substrates are carboxylicacid-firefly luciferin conjugates such as acetyl firefly luciferin. The—COOH group of the carboxylic acid are coupled with the 6-OH group ofthe firefly luciferin to generate an ester bond. Suitable carboxylicacids include, but are not limited to, alkyl substituted carboxylic acidsuch as formic acid, acetic acid, propionic acid, butyric acid and etc.as well as aromatic acid such as benzoic acid. The esterase hydrolyzesthe ester bond of the conjugate to release free firefly luciferin, whichbecomes a substrate for luciferase in a biochemiluminescent reactionthat generates a light signal.

In certain embodiments, the substrate are the firefly luciferin-alcoholconjugates, which is the ester formed by coupling the —COOH group offirefly luciferin with the —OH of an alcohol, resulting in, for example,firefly luciferin methyl ester or firefly luciferin ethyl ester. Thealcohol can be either alkyl alcohol such as methanol, glycerol oraromatic alcohol such as benzyl alcohol. The esterase hydrolyzes theester bond of the conjugate to release free firefly luciferin, whichbecomes a substrate for luciferase in a chemiluminescent reaction thatgenerates a light signal. Elevated light signal indicates high LEactivity, which in turn is indicative of an active infection.

Examples of firefly luciferin-carboxylate conjugate and fireflyluciferin-alcohol conjugate are depicted in FIG. 9. Methods forsynthesizing these esters are well known to the skilled in the art.Purification of the ester can also be accomplished using HPLC in afashion similar to the purification strategy described in EXAMPLE 1 forthe purification of N-acetylneuraminic acid-firefly luciferin.Formulation of the detection mix can be derived from the protocoldescribed in EXAMPLE 1 or 2 as well.

For clinical use, again a cutoff value needs to be established bytesting a large number of negative samples, e.g., 100 or more ofnegative samples. Sensitivity of the assay can be determined by testinga large number of positive samples, e.g., 100 or more of positivesamples.

EXAMPLE 9 Chemiluminescent alpha-L-fucosidase (AFU) Assays

The alpha-L-fucosidase (AFU) assay is for the determination of AFUactivity in patient serum samples. AFU is a lysosomal enzyme involved inthe degradation of a diverse group of naturally occurringfucoglycoconjugates. Serum AFU activity is considered a useful marker ofhepatocellular carcinoma (HCC). Elevated AFU levels in serum are anearly indication of HCC. Though measurement of serum fetoprotein (AFP)is a common practice for early detection of HCC, use of AFP assay alonesuffers from its low specificity and sensitivity, due to the fact thatnot all HCC secrete AFP. AFP levels may be normal in as many as 40% ofpatients with early HCC and 15-20% of patients with advanced HCC. Recentstudies have clearly demonstrated that measurements of both AFP and AFUcan significantly increase the detection specificity and sensitivity forHCC. AFU is reported to be a more sensitive marker, especially fordetecting a small tumor size of HCC. It has also been shown thatabnormal AFU level exists in serum samples from patients suffering fromadult leukemia or ovarian carcinoma. In addition, AFU level is also highin patients suffering from liver cirrhosis and chronic hepatitis.

According to the present invention, AFU level in a sample is quantifiedusing a synthetic substrate alpha-L-fucopyranoside-firefly luciferinconjugate, whose chemical structure is best understood by referring toFIG. 3, which depicts a conjugate between neuraminic acid and fireflyluciferin. In the alpha-L-fucopyranoside-firefly luciferin conjugate,the neuraminic acid portion in FIG. 3 is replaced withalpha-L-fucopyranoside. An example of the conjugate is depicted in FIG.10.

In a chemiluminescent AFU assay, alpha-L-fucopyranoside-fireflyluciferin conjugate is cleaved by alpha-L-fucosidase in a sample torelease free firefly luciferin, which can be quantified in achemiluminescence reaction in the presence of firefly luciferase, ATPand other appropriate conditions. It can be a one-step assay or twosteps assay as those described in EXAMPLES 1 and 2.

Synthesis and purification of alpha-L-fucopyranoside-firefly luciferinconjugate can use a protocol similar to that for synthesis andpurification of N-acetylneuraminic acid-firefly luciferin conjugate asdescribed in EXAMPLE 1.

The assay kit can be similar to those used for neuraminidase detectionexcept the substrate is firefly luciferin-alpha-L-fucopyranosideinstead. The detection mix, either one step or two step form, can beformulated according to the formula and protocols described in EXAMPLES1 and 2. The assay protocol can be readily adopted from the influenzaneuraminidase test or bacterial vaginosis test described above by askilled in the art.

EXAMPLE 10 Chemiluminescent GPDA Assays

Elevated glycylproline dipeptidyl aminopeptidase (GPDA) activity inblood and urine is associated with abnormality in liver, stomach,intestine and kidney. Elevated GPDA activity (especially the isoenzyme,GPDA-F) in serum has been identified as a reliable marker enzyme forhepatocellular carcinoma and other liver disease.

The GPDA assay in the current invention is based on the enzymaticcleavage of the synthetic substrate L-glycyl-L-prolyl-6-amino fireflyluciferin (FIG. 11), whose chemical structure is similar to that ofL-prolyl-6-amino firefly luciferin. In the L-glycyl-L-prolyl-6-aminofirefly luciferin conjugate, the peptide moiety is L-glycyl-L-proline.Synthesis and purification can also be accomplished using methodssimilar to those for L-prolyl-6-amino firefly luciferin conjugate.

In the GPDA assay, the L-glycyl-L-prolyl-6-amino firefly luciferinconjugate is cleaved by GPDA to give rise to free luciferin, which isdetected in a firefly luciferase catalyzed chemiluminescence reaction.It can be a one-step assay or two steps assay as described in Examples 1and 2. Reagent and kit formulation can be similar to those described inExamples 1 and 2 as well.

For clinical use, again a cutoff value can be established by testing alarge number of negative clinical samples, e.g., 100 or more of negativesamples. Sensitivity of the assay can be determined by testing a largenumber of positive clinical samples, e.g., 100 or more of positivesamples. The positive samples are those from patients who are confirmedpositive for hepatocellular carcinoma (HCC) with other well-recognizedmethods.

EXAMPLE 11 Chemiluminescent Assays for Candida Yeast Detection

Many of the yeast Candida species, particularly Candida albican, containa unique enzyme, beta-galactosaminidase. The Candida species,particularly Candida albican, are the most common pathogens that causeyeast vaginal infection. Thus, a substrate for beta-galactosaminidasewould be useful for detecting Candida species infection.

The suitable substrates for firefly luciferase catalyzedbiochemiluminescence reaction is a conjugate betweenbeta-galactosaminidase and firefly luciferin (fireflyluciferin-N-acetyl-beta-D-galactosaminide), which are linked togetherthrough an appropriate chemical bond that can be cleaved bybeta-galactosaminidase. Example of a substrate is shown in FIG. 12.

In the presence of beta-galactosaminidase in a sample, the conjugate iscleaved to release free firefly luciferin, which is detected in afirefly luciferase catalyzed biochemiluminescence reaction. Formulationsof the reagents and test kits and detection procedures can be adoptedfrom those described in Examples 1 and 2 for neuraminidase detectionusing N-acetylneuraminic acid-firefly luciferin conjugate. Again, alarge number of clinical samples need to be tested to established thecutoff value for a yeast infection, e.g., vaginal yeast infection.

In addition to beta-galactosaminidase, Candida albicans producesL-proline aminopeptidase. Thus, to further differentiate the infectionbetween Candida albicans and other Candida species, both enzymes can beassayed. A biochemiluminescence substrate for L-proline aminopeptidaseand its use for L-proline aminopeptidase detection are described inEXAMPLE 7. A sample with elevated activities for both enzymes isconsidered positive for Candida albicans infection. The cutoff valuesfor both enzymes can be established by testing a large number, e.g.,100, of negative samples and a large number, e.g., 100, of positivesamples.

Formulations of the reagents and test kits and detection procedures canbe adopted from Examples 1 and 2 for neuraminidase detection usingN-acetylneuraminic acid-firefly luciferin conjugate.

EXAMPLE 12 A Chemiluminescent Assay for Lipid Esterase and SalmonellaSpecies Detection

The Salmonella esterase catalyzes the hydrolysis of a variety of C6 toC16 fatty acid esters but does not hydrolyze peptide bonds. Presence ofthis esterase activity is indicative of Salmonella species presence in asample.

The current invention relates to novel chemiluminescent C6 to C16 fattyacid ester esterase substrates that can be used to detect Salmonella.The Salmonella esterase can cleave the substrates and release thechemiluminescent moiety firefly luciferin, which is detected in aluciferase-catalyzed reaction. The substrates useful for the currentinvention are firefly luciferin fatty acid ester (C6 to C16 fatty acid).The —COOH group of the fatty acid are coupled with the 6-OH group of thefirefly luciferin to generate an ester. An example of thus an ester isdepicted in FIG. 13. One preferred fatty acid is caprylic acid.

The synthesis of firefly luciferin based Salmonella esterase substratescan be derived from a published article or articles or text book thatprovides a protocol for the synthesis of a lipid ester. For example, thesynthesis can be performed by incubating the firefly luciferin withexcessive amounts of C6 to C16 fatty acid chloride (e.g., caprylylchloride) in pyridine at room temperature for 1 hour, followed by silicagel column purification to remove the unreacted reactants (e.g., thefatty acid). The recovered firefly luciferin fatty acid ester will befurther purified using a semi-preparative HPLC to remove the unreactedfirefly luciferin and fatty acid.

EXAMPLE 13 A Chemiluminescent Assay for Hydroxyproline AminopeptidaseDetection

Vaginal samples from Neisseria gonorrhoeae patients show high level ofhydroxyproline aminopeptidase activity. Therefore, hydroxyprolineaminopeptidase assay can be used to detect Neisseria gonorrhoeaeinfection. Hydroxyproline-firefly luciferin, depicted in FIG. 14, can bea substrate for detection of the enzyme and therefore, diagnosis ofNeisseria gonorrhoeae infection.

Methods for substrate synthesis, purification and test kit formulationare similar to those used for praline aminopeptidase (Example 7) and tothose for neuraminidase (Examples 1 and 2).

The preceding examples can be repeated with similar success bysubstituting the generically or specifically described reactants and/oroperating conditions of this invention for those used in the precedingexamples.

From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this invention and, withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions.

1. A compound of formula I

wherein R₁-R₃ are each, independently of one another, H or C₁₋₅ alkylhaving 1-5 carbon atoms, or a salt thereof.
 2. The compound of claim 1,wherein said alkyl is a straight chain or a branched C₁₋₅ alkyl having1-5 carbon atoms.
 3. The compound of claim 1, wherein said alkyl ismethyl.
 4. A composition comprising a compound of claim 1 and a chemicalcompatible with a (bio) chemiluminescence reaction.
 5. A kit comprisinga compound of claim 1 and a buffer in one or more packages.
 6. The kitaccording to claim 5 which further comprises firefly luciferase.
 7. Thekit according to claim 6 which further comprises ATP.
 8. The kitaccording to claim 6 which additionally comprises a neuraminidaseinhibitor in a separate package.
 9. A method for detecting neuraminidasein a sample, comprising: (a) contacting said sample with a compound ofclaim 1 under conditions effective for the compound to be cleaved byneuraminidase to release firefly luciferin; and (b) detecting thereleased firefly luciferin.
 10. The method according to claim 9, whereinsaid released firefly luciferin is detected via a biochemiluminescencereaction in the presence of firefly luciferase.
 11. The method accordingto claim 9, wherein said neuraminidase is viral or bacterialneuraminidase.
 12. A method for detecting influenza virus in a sample,comprising: (a) contacting said sample with a compound of claim 1 underconditions effective for the compound to be cleaved by influenza virusneuraminidase to release firefly luciferin; and (b) detecting thereleased firefly luciferin.
 13. The method according to claim 12,wherein said released firefly luciferin is detected via abiochemiluminescence reaction in the presence of firefly luciferase. 14.The method according to claim 12, wherein said neuraminidase is viral orbacterial neuraminidase.
 15. A method for diagnosing influenza virusinfection in a subject in need thereof comprising (a) isolating abiological sample from said subject: (b) contacting said sample with acompound of claim 1 under conditions effective for the compound to becleaved by influenza virus neuraminidase to release firefly luciferin;and (c) detecting the released firefly luciferin.
 16. The methodaccording to claim 15 wherein said influenza virus is type A or type Binfluenza virus.
 17. The method according to claim 15 wherein saidsubject is a human patient or a veterinary animal.